Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM

Alexander G. Georgiev, David P. Sullivan, Michael C. Kersting, Jeremy S. Dittman, Christopher T. Beh, Anant K. Menon*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

88 Scopus citations

Abstract

Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP-dependent, non-vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologs of the mammalian oxysterol-binding protein (Osh1-7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM-derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔosh4-1 ts) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse-chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM-enriched fractions as well as by extracting sterols from intact cells with methyl-β-cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh-deficient cells, the rate at which it moves within the PM to equilibrate with the methyl-β-cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in non-vesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM.

Original languageEnglish (US)
Pages (from-to)1341-1355
Number of pages15
JournalTraffic
Volume12
Issue number10
DOIs
StatePublished - Oct 2011

Keywords

  • Cholesterol
  • Cyclodextrin
  • Dehydroergosterol
  • Detergent-resistant membrane
  • Endoplasmic reticulum
  • Ergosterol
  • Lipid droplet
  • Lipid transfer protein
  • Non-vesicular transport
  • Osh protein
  • Oxysterol-binding proteins
  • Plasma membrane
  • Yeast

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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